The present invention relates to nucleotide sequences encoding a family of mammalian calcium activated chloride channels which may alternatively, or additionally function as adhesion molecules. More particularly, the invention is directed to genes isolated from bovine endothelial cells, human At endothelial cells and murine endothelial cells, which encode calcium activated chloride channel molecules and include the lung-endothelial cell adhesion molecules (Lu-ECAM-1) and associated proteins.
Calcium Activated Chloride Channels
Ion channels are not only required for normal cellular functions but also play a critical role in numerous diseased states. For example, cystic fibrosis results when ion transport in epithelial cells of individuals is altered due to a genetic defect of the cystic fibrosis transmembrane conductance regulator CFTR; Knowles et al., 1983, J. Clin. Invest.71:1410-1417). Although serious airway pathology is usually the primary cause of mortality in young adults with CF, intestinal epithelial alterations have also been observed. However, the severity of tissue lesions does not correlate with the expression of CFTR in humans or mice, suggesting the involvement of cell-specific channels in addition to CFTR. Further support for the involvement of other channel protein molecules in CF comes from observations that calcium activated chloride secretion is preserved in respiratory epithelia of CF patients compared to unaffected individuals, but is significantly reduced or absent from CFTR-defective epithelia. These results strongly suggest that an alternative non-CFTR regulated chloride channel activity might account for attenuating CF disease in some tissues. Thus, a need exists for identification, isolation and functional analysis of alternative chloride channels.
Adhesion Molecules
It is apparent that endothelial cell adhesion molecules may have functions in addition to their adhesive functions. For example, integrins have transmembrane signaling capacities which may play a role in the adherence process. However, the primary function of endothelial cell adhesion molecules is adherence to a substrate such as (a) to promote adherence of endothelial cells to basement membrane, (b) to promote vascular arrest and to facilitate extravasation of leukocytes such as during an immune response, and (c) to promote homing of lymphocytes to a particular lymphoid tissue. Other molecules may play a role in controlling adherence of endothelial cells. For example, chloride ion channels are thought to be involved in a signaling cascade when lymphatic endothelial cells begin to adhere to a substrate (Martin et al., 1996, Microvasc. Res. 52:200-9).
There is considerable evidence that metastatic nonlymphoid tumor cells mimic leukocytes in recognizing and adhering to one or more endothelial cell adhesion molecules to migrate in blood vessels, to arrest in vascular areas of organs which may provide the microenvironment conducive for metastatic growth, and to extravasate into surrounding tissues. An example of such an endothelial cell adhesion molecule which promotes adhesion of tumor cells and mediates metastasis is lung-endothelial cell adhesion molecule (Lu-ECAIVI-1). Lu-ECAM-1 is a 90 kilodalton (kDa) integral membrane protein constitutively expressed primarily in endothelial cells of pleural and subpleural microvessels. Both in vitro studies and in vivo studies indicate that Lu-ECAM-1-expressing endothelial cells promote adhesion of certain lung-colonizing tumor cells in a manner that is consistent with the expression level of the adhesion molecule and the metastatic propensity of tumor cells. For example, in an in vitro tumor cell/endothelial cell adhesion assay, highly lung metastatic B1G-F10 melanoma cells bind to lung-matrix-modulated endothelial cells expressing Lu-ECAM-1 in significantly larger numbers than their intermediate or low lung-metastatic counterparts (B1G-L8-F1O and B1GFO, respectively; Zhu et al., 1991, Proc. Nati. Acad. Sd. USA 88:9568-720). Such binding appears to be calcium (Ca2+) dependent. Further, anti-Lu-ECAM-1 monoclonal antibodies significantly inhibit adhesion of B1GF10 melanoma cells to Lu-ECAM-1 expressing endothelial cells in culture (Zhu et al., 1991, supra) Anti-Lu-ECAM-1 monoclonal antibodies are also efficient in preventing metastatic colonization of the lungs by highly lung-metastatic B1GF10 cells in a standard animal model for metastasis (Zhu et al., 1991, supra). Lu-ECAM-1, affinity purified from detergent extracts of bovine aortic endothelial cells, was used to immunize mice. The immunized mice showed an inhibition of metastatic colonization of the lungs by B1GF10 melanoma cells, the efficiency of which was dependent upon the anti-Lu-ECAN-l serum titer (Zhu et al., 1992, J. Clin. Invest. 89:1718-1724). Lu-ECAM-1 appears to be the endothelial cell adhesion molecule for metastatic tumor cells that express the ligand/34 integrin subunit (and possibly other ligands) including, but not limited to, lung-metastatic breast tumor cells, and lung-metastatic melanoma tumor cells.
Anti-adhesion therapy may be used to interfere with adhesion between organ-specific endothelial cells and blood-borne cancer cells in preventing the formation of metastatic colony formation in organs that support metastatic cell growth. The amount of endothelial cell adhesion molecule that can be made from detergent extracts, as well as the rate of production of the endothelial cell adhesion molecule, is generally insufficient for cost-effective commercial production. More efficient production of proteins, with a concomitant reduction in production cost, can often be achieved by producing a protein through recombinant means. In that regard, in some cases a host cell may be genetically engineered such that an increased amount of the protein is produced and/or the protein is produced in a manner which facilitates its isolation (as compared to harvesting the protein from cell membranes).
It is an object of the invention to provide nucleotide sequences, isolated from mammalian endothelial cells, which encode molecules that functions as a calcium activated chloride channel-adhesion molecule (CACC-AM).
It is also an object of the present invention to provide nucleotide sequences which are variants (including portions) of the gene comprising the CACC-AM, and which encode a polypeptide having substantially the biological activity as compared to the biological activity of the CACC-AM.
It is an object of the present invention to provide a means for recombinantly producing CACC-AM molecule.
It is an object of the present invention to provide a means for recombinantly producing proteins associated with CACC-AM molecule.
It is a further object of the present invention to provide expression vectors containing a nucleotide sequence that encodes a CACC-AM molecule; or containing a nucleotide sequence which is a variant of the gene for CACC-AM, and that encodes a polypeptide having substantial biological activity of a CACC-AM; or containing a nucleotide sequence that encodes a protein associated with a CACC-AM.
It is an additional object of the present invention to provide recombinant host cells which contain multiple copies of a nucleotide sequence that encodes a CACC-AM molecule, wherein the CACC-AM molecule is recombinantly produced by culturing the recombinant host cells under suitable conditions.
Other objects, features, and advantages of the present invention will become apparent from the following drawings and detailed description.